Functional studies of HIF-1α and HIF-2α are complicated by the fact that most cell lines express both HIF-1α and HIF-2α. Elimination of HIF-1α expression in Hif-1α−/−
ES cells and HIF-2α expression in Hif-2α−/−
ES cells provides us with a good opportunity to investigate the relative contributions of HIF-1α and HIF-2α to hypoxic responses. Results from previous studies indicate that HIF-2α in Hif-1α−/−
ES cells does not regulate a number of glycolytic genes (4
). Recently, we and others found that glycolytic genes are preferentially regulated by HIF-1α (16
). Thus, HIF-2α's function in ES cells was unclear. We performed DNA microarray analysis of hypoxia-inducible genes in WT, Hif-1α−/−
, and Hif-2α−/−
ES cells and identified 60 identical hypoxia-inducible genes in either WT or Hif-2α−/−
ES cells. The majority of those tested were confirmed by Northern blot analysis. Besides several known hypoxia-inducible genes, a large number of novel hypoxia-inducible genes, such as Upp
, and Flnb
, were also O2
regulated in the WT and Hif-2α−/−
ES cells (Table ). Induction in the HIF-1α-expressing WT and Hif-2α−/−
ES cells but not in Hif-1α−/−
ES cells suggests they are true HIF target genes.
Our DNA microarray study clearly shows that HIF-1α regulates a large number of target genes in ES cells. However, to our surprise, HIF-2α is not functional in ES cells. This conclusion is based on several lines of evidence. First, we determined that Hif-1α−/− ES cells express full-length HIF-2α mRNA and are devoid of HIF-2α isoforms (Fig. ). Next, we demonstrated that Hif-1α−/− ES cells exhibit no HIF-mediated hypoxic gene induction by investigating endogenous HIF-2α target genes as well as more-sensitive HRE reporters under a variety of stimuli, including severe hypoxia and several hypoxia mimetics. In addition, low levels of induction of several genes, including Vegf, Anaxa-2, and Ldha, observed in Hif-1α−/− ES cells is due to HIF-independent regulation, since similar induction is observed in Arnt−/− ES cells. Finally, HIF-2α lacks transcriptional activity in ES cells. If all the hypoxia-inducible genes observed in the WT or Hif-2α−/− ES cells were HIF-1α unique target genes, then we could not conclude that HIF-2α is inactive here. We previously showed that HIF-2α regulates genes such as Vegf, Glut-1, Adrp, Adm, and Ndrg-1 in 786-O cells, and these genes are also induced by HIF-1α in ES cells, as shown in Fig. and Table , indicating they are HIF-1α/HIF-2α common target genes. Indeed, these genes can be induced by HIF-2α when HIF-2α becomes functional in ES cells (Fig. ), while the HIF-1α unique target genes Pgk and Ldha remain unresponsive to overexpressed HIF-2α in ES cells (data not shown). Induction of the HIF-1α/HIF-2α common target genes Vegf, Glut-1, Adrp, Adm, and Ndrg-1 exclusively by HIF-1α but not by HIF-2α demonstrates that endogenous HIF-2α does not regulate its target genes in ES cells.
Having confirmed that the WT HIF-2α protein is expressed and binds to target gene promoters, we hypothesized that HIF-2α inhibition in ES cells occurs at the step of transcriptional cofactor recruitment. Although ES cells express levels of the HIF-2α protein similar to or even higher than that expressed by 786-O WT8 cells (as determined using an antibody generated from a peptide conserved between human and mouse HIF-2α) (Fig. ), this endogenous level of the HIF-2α protein is sufficient to induce target gene expression in 786-O cells but not in Hif-1α−/− ES cells. This difference suggests either that ES cells express a HIF-2α-specific transcriptional repressor or that they lack a HIF-2α-specific activator. Restoration of HIF-2α function in ES cells by overexpressed WT HIF-2α, particularly by the HIF-2α mutant, but not by the similar HIF-1α mutant suggests the presence of a HIF-2α-specific transcriptional repressor in these cells. Partial restoration of endogenous HIF-2α function by the HIF-2α mutant is likely due to its having both a positive effect in relieving repressor binding to endogenous HIF-2α and a negative effect by sequestering other transcriptional coactivators from endogenous HIF-2α. This experiment also proves that ES cells produce a functional HIF-2α transcript. Based on these data, we propose the following model: in ES cells, HIF-2α/ARNT dimers occupy target gene HREs but fail to activate transcription due to the expression of a HIF-2α-specific transcriptional corepressor. We hypothesize that this repressor inhibits the function of HIF-2α in ES cells by preventing HIF-2α from interacting with general transcriptional factors on the promoters. Overexpressed WT or a DNA-binding-defective HIF-2α protein serves as a sink for the repressor, allowing the repressor-free HIF-2α protein to stimulate target gene expression (Fig. ). While our experiments collectively provided strong evidence for a repressor, definitive proof will ultimately come from the “rescue” of HIF-2α function in ES cells upon siRNA knock-down of the repressor.
FIG. 10. Proposed model for HIF-2α inhibition in ES cells. (A) HIF-2α/HIF-1β(ARNT) dimers bind target gene HREs but fail to stimulate their transcription, likely due to inhibition of HIF-2α interaction with general transcription (more ...)
The HIF-1α and HIF-2α proteins exhibit a high degree of homology in their bHLH and PAS regions, domains important for DNA binding and ARNT dimerization. However, the two proteins exhibit limited similarities in their C termini, particularly the inhibitory domains and N-terminal transactivation domains. The structural diversity provides a molecular basis for their unique transcriptional cofactor requirements. For example, the NF-κB essential modulator (NEMO) has been shown to exclusively interact with HIF-2α and to promote its transcriptional activity by enhancing binding to CBP/p300 (3
). NEMO is a coactivator for HIF-2α and is expressed in ES cells (according to DNA microarray data), indicating that NEMO is not the explanation for lack of HIF-2α function in ES cells. Another example of a HIF-2α-specific regulator is a proposed cytoplasmic protein in MEFs that prevents HIF-2α translocation to the nucleus (35
). However, a recent report indicates that HIF-2α is not expressed in some MEF isolates (3
). We also failed to detect HIF-2α protein in our primary MEF cultures (data not shown). Since HIF-2α in ES cells exhibits normal pVHL regulation and nuclear translocation, the putative HIF-2α repressor in ES cells is different from that in MEFs. CITED-2 and its isoform p35srj have been shown to inhibit transcriptional activity of HIF-1α by preventing coactivators p300/CBP and SRC-1 from binding to the HIF-1α C-terminal transactivation domain (2
). We determined that p300, SRC-1, and CITED-2 are expressed in ES cells (see Fig. S2 in the supplemental material). Expression of CITED-2 might explain HIF-2α inactivity in ES cells if CITED-2 has stronger inhibitory effects on HIF-2α. However, transfection of a CITED-2 expression plasmid did not prevent either HIF-1α and HIF-2α induction of cotransfected HRE-mediated reporter genes in ES cells, although CITED-2 inhibited HIF-1α and HIF-2α similarly in HEK293 cells (see Fig. S2 in the supplemental material). Thus, misexpression of coactivators p300 and SRC-1 or expression of CITED-2 is not involved in HIF-2α's inability to regulate its target genes in ES cells.
ChIP assays demonstrate that endogenous HIF-2α/ARNT dimers bind to the HREs of the HIF target genes Glut-1
. Interestingly, the binding of HIF-1α (or HIF-2α) to the HREs is not enhanced by deletion of its counterpart α subunit in ES cells, suggesting that HIF-1α and HIF-2α might not bind the identical DNA elements. This is in accordance with the fact that HREs from Glut-1
contain at least two potential binding sites, a conventional HIF-binding site (CGTG) and an inverted repeat (CACG for Glut-1
and CAGG for Vegf
). It is worth noting that the HIF-1α protein induces Glut-1
expression to similar levels in both Hif-2α−/−
and WT ES cells, consistent with the idea that HIF-2α DNA binding in WT ES cells does not interfere with HIF-1α function. A more direct assay, such as in vivo DNA footprinting, is required to determine whether the above-described phenomenon is the consequence of unsaturated binding or occupying unique HIF binding sites on the promoters.
The discovery that some HIF-2α-expressing cells may not harbor a functional HIF-2α protein helps to resolve some confusion about the role of HIF-2α in hypoxic responses. It is now clear that loss of HIF-1α abolishes all HIF-dependent hypoxic gene induction in ES cells, since HIF-2α is not functional in these cells. Furthermore, our data also indicate that expression of HIF-2α mRNA and protein does not guarantee HIF-2α activity; thus, a meaningful study of target gene specificity using siRNA technology to specifically knock down HIF-1α or HIF-2α must use cells where both HIF-1α and HIF-2α are active.
The tumor-promoting effects of VHL
mutations appear to be dependent on HIF-2α-mediated target gene regulation. Thus, lack of HIF-2α activity might block this effect of VHL
mutations. HIF-2α has been shown to play a critical role in tumor formation by pVHL-deficient RCC cells, and HIF-2α is clearly functional in these cells (16
). Nonfunctional HIF-2α in ES cells might be partially responsible for our observation that pVHL-deficient ES cells fail to promote tumor growth in nude mice, despite the fact that these ES cells display constitutive HIF-1α and HIF-2α protein and HIF-1α target gene expression, as observed for pVHL-deficient RCC cells (29
). In agreement with the teratoma model, fibrosarcomas derived from Vhl−/−
MEFs are significantly smaller than WT controls (28
), and MEFs appear to harbor no HIF-2α activity due to cytoplasmic trapping (35
) or lack of expression (3
). In contrast to Vhl−/−
ES cells, ES cell-derived teratomas with targeted replacement of HIF-1α by HIF-2α display a proliferative advantage in comparison to WT ES cells (8
). Increased teratoma expression of HIF-2α target genes, such as Vegf
, transforming growth factor α (TGF-α
), and cyclin D1, suggests that HIF-2α is functional in these cells, presumably by overcoming a HIF-2α repressor through increased expression of the HIF-2α protein. Although VHL
are broadly expressed, human VHL
mutations give rise to only a few tissue-specific tumors. Expression of a HIF-2α-specific repressor might play a role in limiting the tissue penetration of VHL mutations. It will be interesting to identify the HIF-2α repressor and to see whether there is a negative correlation between tissues that express the HIF-2α repressor and risk levels of VHL disease.
Recently, we and others showed that HIF-2α regulates TGF-α
and the Pou transcription factor Oct-4
(also known as Oct-3/4
) in RCC cells (16
). Moreover, these genes are specifically regulated by HIF-2α in RCC cells (38
). Since HIF-2α is expressed and its protein is stabilized in hypoxic ES cells, the inhibition of HIF-2α target gene expression by a HIF-2α transcriptional repressor may be required for proper ES cell function. Expression of Oct-4
in undifferentiated ES cells is essential for maintaining stem cell pluripotency (33
). When Oct-4 expression is reduced or increased, ES cells lose pluripotency and start differentiating, demonstrating the importance of maintaining correct Oct-4 expression levels (34
). We recently observed that expanded HIF-2α expression in ES cells by HIF-2α knock-in at the Hif-1α
locus leads to severe developmental defects in mouse embryos, defective hematopoetic stem cell differentiation in embryoid bodies, and larger teratomas (K. L. Covello et al., submitted for publication). Interestingly, we found that enhanced Oct-4
expression in these cells is partially responsible for these defects, providing strong evidence for the tight regulation of HIF-2α activity in ES cells and early embryonic development.
Our results demonstrated that HIF-2α is not functional in some cell types, such as ES cells. However, HIF-1α appears to be functional in all cells tested. This might reflect their distinct expression patterns and functions. For example, HIF-1α is present in organisms from Caenorhabditis elegans
to humans, while HIF-2α exists only in more complicated vertebrate species, such as chicken, quail, and mammals (9
). In addition, HIF-1α is universally expressed while HIF-2α expression is more tissue restricted, even in organisms where both isoforms are expressed. From a functional standpoint, HIF-1α appears to regulate genes involved in basic cellular activity, like the glycolytic pathway, while HIF-2α is more involved in genes having special function such as Oct-4
In summary, we demonstrate that HIF-1α, but not HIF-2α, is functional in ES cells, showing a clear regulatory difference between HIF-1α and HIF-2α. While hypoxic stabilization of HIF-α is a critical regulatory step for HIF transcriptional activity, stabilization of HIF-2α is insufficient for transcriptional activity. Furthermore, we provide compelling evidence for a model whereby HIF-2α's inability to regulate its target genes in ES cells is due to the expression of a HIF-2α-specific corepressor that globally inactivates its function. Identification of such a novel regulatory mechanism is critical to understanding the role of HIF-2α in hypoxic responses and in tumorigenesis and provides a possible explanation for VHL disease tissue specificity.